Oral immunization of pigs against classical swine fever. Course of the disease and virus transmission after simultaneous vaccination and infection

The efficacy of simultaneous vaccination of pigs against classical swine fever (CSF) and challenge was evaluated. In this study, domestic weanling pigs were vaccinated orally with a conventional live virus vaccine based on CSF virus (CSFV) C strain and were challenged simultaneously with CSFV of different virulence. All the animals vaccinated and challenged with a high dose of highly virulent Koslov strain died while three of five animals challenged with a low dose of highly virulent Alfort 187 strain survived, shed the virus in nasal secretions, developed antibodies, and four of them showed a transient viremia. All the animals vaccinated and challenged with the low virulent field isolate MV 140/Riems survived, showed a short viremia and developed antibodies. No CSFV or CSFV RNA could be detected in the animals surviving the infection. This study demonstrates that oral vaccination of wild boars in an infected area bears no risk for the development of a persistent CSF infection.     

Role of double-stranded RNA and Npro of classical swine fever virus in the activation of monocyte-derived dendritic cells

Classical swine fever virus (CSFV) is a noncytopathogenic (ncp) positive-sense RNA virus that replicates in myeloid cells including macrophages and dendritic cells (DC). The virus does not induce type I interferon (IFN-alpha/beta), which in macrophages has been related to the presence of the viral Npro gene. In the present work, the role of viral double-stranded (ds)RNA and Npro in the virus-host cell interaction has been analyzed. Higher levels of detectable dsRNA were produced by a genetically engineered cytopathogenic (cp) CSFV compared with ncp CSFV, and cp CSFV induced IFN-alpha/beta in PK-15 cells. With DC, there was only a small difference in the levels of dsRNA between the cp and ncp viruses, and no IFN-alpha/beta was produced. However, the cp virus induced a higher degree of DC maturation, in terms of CD80/86 and MHC II expression. Npro deletion mutants induced an increase in DC maturation and IFN-alpha/beta production-for both ncp and cp viruses-despite reduced replication efficiency in the DC. Deletion of Npro did not influence dsRNA levels, indicating that the interference was downstream of dsRNA turnover regulation. In conclusion, the capacity of CSFV to replicate in myeloid DC, and prevent IFN-alpha/beta induction and DC maturation, requires both regulated dsRNA levels and the presence of viral Npro.     

Evaluation of a real-time RT-PCR assay using minor groove binding probe for specific detection of Chinese wild-type classical swine fever virus

A one-step real-time RT-PCR assay using a minor groove binding probe was developed for the specific detection of Chinese wild-type classical swine fever virus (CSFV). The assay detected wild-type CSFV strains representing different genotypes, but did not amplify viral RNA from the Hog Cholera Lipinized Virus (HCLV) vaccine-strain and other porcine viruses. The assay had a detection limit of 10 copies/reaction or 3.0 median tissue culture infective dose/reaction. In comparison to the sequencing nested RT-PCR assay, the sensitivity and specificity of the assay were 98.3% and 94.3%, respectively, when testing 515 veterinary samples. Wild-type CSFV RNA was detected in nasal swabs 2-4 days before detection in serum samples from pigs exposed to infection by contact, and 2-4 days prior to the onset of clinical disease. HCLV RNA remained undetectable in nasal swabs and serum samples from vaccinated pigs. In conclusion, the novel assay described in this study provides a rapid and sensitive method for differentiating between wild-type and the HCLV-strain of CSFV. It could be used for monitoring in CSF outbreak areas or as a screening method for CSFV eradication strategies.     

Identification of an NTPase motif in classical swine fever virus NS4B protein

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by CSF virus (CSFV), a positive-sense single-stranded RNA virus within the Pestivirus genus of the Flaviviridae family. Here, we have identified conserved sequence elements observed in nucleotide-binding motifs (NBM) that hydrolyze NTPs within the CSFV non-structural (NS) protein NS4B. Expressed NS4B protein hydrolyzes both ATP and GTP. Substitutions of critical residues within the identified NS4B NBM Walker A and B motifs significantly impair the ATPase and GTPase activities of expressed proteins. Similar mutations introduced into the genetic backbone of a full-length cDNA copy of CSFV strain Brescia rendered no infectious viruses or viruses with impaired replication capabilities, suggesting that this NTPase activity is critical for the CSFV cycle. Recovered mutant viruses retained a virulent phenotype, as parental strain Brescia, in infected swine. These results have important implications for developing novel antiviral strategies against CSFV infection.     

Human MxA protein inhibits the replication of classical swine fever virus

Classical swine fever virus (CSFV) has a spherical enveloped particle with a single stranded RNA genome, the virus belonging to a pestivirus of the family Flaviviridae is the causative agent of an acute contagious disease classical swine fever (CSF). The interferon-induced MxA protein has been widely shown to inhibit the life cycle of certain RNA viruses as members of the Bunyaviridae family and others. Interestingly, it has been reported that expression of MxA in infected cells was blocked by CSFV and whether MxA has an inhibitory effect against CSFV remains unknown to date until present. Here, we report that CSFV replicated poorly in cells stably transfected with human MxA. The proliferation of progeny virus in both PK-15 cell lines and swine fetal fibroblasts (PEF) continuously expressing MxA was shown significantly inhibited as measured by virus titration, indirect immune fluorescence assay and real-time PCR.     

Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins

Classical swine fever virus (CSFV) harbors three envelope glycoproteins (E^rns, E1 and E2). Previous studies have demonstrated that removal of specific glycosylation sites within these proteins yielded attenuated and immunogenic CSFV mutants. Here we analyzed the effects of lack of glycosylation of baculovirus-expressed E^rns, E1, and E2 proteins on immunogenicity. Interestingly, E^rns, E1, and E2 proteins lacking proper post-translational modifications, most noticeable lack of glycosylation, failed to induce a detectable virus neutralizing antibody (NA) response and protection against CSFV. Similarly, no NA or protection was observed in pigs immunized with E1 glycoprotein. Analysis of E^rns and E2 proteins with single site glycosylation mutations revealed that detectable antibody responses, but not protection against lethal CSFV challenge is affected by removal of specific glycosylation sites. In addition, it was observed that single administration of purified E^rns glycoprotein induced an effective protection against CSFV infection.     

A multiplex nested RT-PCR for the detection and differentiation of wild-type viruses from C-strain vaccine of classical swine fever virus

A multiplex nested RT-PCR (RT-nPCR) was developed for the detection and differentiation of classical swine fever virus (CSFV). A fragment of 447 or 343 bp was amplified from the genomic RNA of C-strain or virulent Shimen strain, respectively, and two fragments of 447 and 343 bp were simultaneously amplified from the mixed samples of C-strain and Shimen. When detecting several wild-type isolates representative of different subgroups (1.1, 2.1, 2.2, and 2.3) circulating in Mainland China and samples from pigs experimentally infected with Shimen strain, the RT-nPCR resulted in an amplification pattern similar to Shimen. No amplification was achieved for uninfected cells, or cells infected with bovine viral diarrhea virus (BVDV), and other viruses of porcine origin. The RT-nPCR was able to detect as little as 0.04 pg of CSFV RNA. The restrictive fragment length polymorphism (RFLP) demonstrated unique patterns of wild-type viruses and C-strain. Among the 133 field samples, 42 were tested to contain wild-type viruses and 18 showing presence of C-strain. The RT-nPCR can be used to detect and differentiate pigs infected with wild-type CSFV from those vaccinated with C-strain vaccine, thus minimizing the risk of culling vaccinates during outbreaks.     

Comparison of the protective efficacy of recombinant adenoviruses against classical swine fever

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious and often fatal swine disease that is responsible for significant losses to the swine industry worldwide. Previously, we demonstrated that pigs immunized with a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV were protected against virulent CSFV; however, a few pigs showed a short-term fever and occasional pathological changes. To enhance the efficacy of the vaccine, we constructed two recombinant adenoviruses, namely, rAdV-E2UL49, which encodes the CSFV E2 gene fused with the UL49 gene from pseudorabies virus (PRV), and rAdV-optiE2, which expresses the codon-optimized CSFV E2 gene. With these viruses, we performed a comparative immunogenicity trial in rabbits and pigs and compared these recombinant adenovirus vaccines (rAdV-E2UL49 and rAdV-optiE2) with the one containing the wild-type E2 gene (rAdV-E2). In terms of antibody titers, IFN-γ production, lymphocyte proliferation, viral loads and clinical protection from the disease, rAdV-E2UL49 was more immunogenic and protective against C-strain CSFV in rabbits and Shimen strain CSFV in pigs than rAdV-optiE2 and rAdV-E2. Data from this study could assist in making decisions for further development of recombinant adenoviruses as vaccine candidates against CSF.     

Insertion of cellular NEDD8 coding sequences in a pestivirus

For the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP 821, a duplication of the genomic region encoding part of NS2, NS3, NS4A, and part of NS4B together with a nonviral insertion was detected. Further analyses including molecular cloning and sequencing of the putative cellular recombination partner showed that the insertion in CP 821 originated from a bovine mRNA encoding the cellular protein NEDD8, which is 58% identical to ubiquitin. To our knowledge the genome of CP 821 represents the first viral RNA with a NEDD8 coding insertion. Remarkably, the insertion site differs from that described for insertions of ubiquitin. The NEDD8 sequence allows an additional cleavage of the viral polyprotein, whereby an NS3 with an unusual N-terminus is generated. Furthermore, the CP 821-specific genomic alterations were introduced into an infectious noncytopathogenic (noncp) BVDV cDNA clone. After transfection of bovine cells with the respective RNA, a cp virus was recovered. This showed that the NEDD8 coding insertion together with the duplicated viral sequences represents the genetic basis for cytopathogenicity of CP 821. In addition to the recovered cp virus, noncp BVDV rapidly evolved after transfection. This is the first time that a change from the cp to the noncp phenotype was demonstrated in the course of replication in tissue culture cells.     

An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus

A chimeric Pestivirus was constructed using an infectious cDNA clone of bovine viral diarrhea virus (BVDV) [J. Virol. 70 (1996) 8606]. After deletion of the envelope protein E2-encoding region, the respective sequence of classical swine fever virus (CSFV) strain Alfort 187 was inserted in-frame resulting in plasmid pA/CP7_E2alf. After transfection of in vitro-transcribed CP7_E2alf RNA, autonomous replication of chimeric RNA in bovine and porcine cell cultures was observed. Efficient growth of chimeric CP7_E2alf virus, however, could only be demonstrated on porcine cells, and in contrast to the parental BVDV strain CP7, CP7_E2alf only inefficiently infected and propagated in bovine cells. The virulence, immunogenicity, and "marker vaccine" properties of the generated chimeric CP7_E2alf virus were determined in an animal experiment using 27 pigs. After intramuscular inoculation of 1 x 10(7) TCID(50), CP7_E2alf proved to be completely avirulent, and neither viremia nor virus transmission to contact animals was observed; however, CSFV-specific neutralizing antibodies were detected from day 11 after inoculation. In addition, sera from all animals reacted positive in an E2-specific CSFV-antibody ELISA, but were negative for CSFV-E(RNS)-specific antibodies as determined with a CSFV marker ELISA. After challenge infection with highly virulent CSFV strain Eystrup, pigs immunized with CP7_E2alf were fully protected against clinical signs of CSFV infection, viremia, and shedding of challenge virus, and almost all animals scored positive in a CSFV marker ELISA. From our results, we conclude that chimeric CP7_E2alf may not only serve as a tool for a better understanding of Pestivirus attachment, entry, and assembly, but also represents an innocuous and efficacious modified live CSFV "marker vaccine".     

Evidence of natural recombination in classical swine fever virus

Classical swine fever (CSF) virus, one member of the family Flaviviridae is the pathogen of CSF, an economically important and highly contagious disease of pigs. Although homologous recombination has been demonstrated in many other members of the family, it is unknown whether there is recombination in natural populations of CSFV. To detect possible recombination events, we performed a phylogenetic analysis of 25 full-length CSFV strains isolated all over the world. Putative recombinant sequences were identified with the use of SimPlot program. Recombination events were confirmed by bootscaning. A mosaic virus, CSFV 39 (AF407339) isolated in China was found. And its two putative parental-like strains CSFV Shimen (AF333000) and GXWZ02 (AY367767) were identified. Our work revealed that homologous recombination occurred in natural CSFV populations, generating genetic diversity. This would provide some insights for the role homologous recombinant plays in CSFV evolution.     

Mutation of E1 glycoprotein of classical swine fever virus affects viral virulence in swine

Transposon linker insertion mutagenesis of a full-length infectious clone (IC) (pBIC) of the pathogenic classical swine fever virus (CSFV) strain Brescia was used to identify genetic determinants of CSFV virulence and host range. Here, we characterize a virus mutant, RB-C22v, possessing a 19-residue insertion at the carboxyl terminus of E1 glycoprotein. Although RB-C22v exhibited normal growth characteristics in primary porcine macrophage cell cultures, the major target cell of CSFV in vivo, it was markedly attenuated in swine. All RB-C22v-infected pigs survived infection remaining clinically normal in contrast to the 100% mortality observed for BICv-infected animals. Comparative pathogenesis studies demonstrated a delay in RB-C22v spread to, and decreased replication in the tonsils, a 10(2) to 10(7) log10 reduction in virus titers in lymphoid tissues and blood, and an overall delay in generalization of infection relative to BICv. Notably, RB-C22v-infected animals were protected from clinical disease when challenged with pathogenic BICv at 3, 5, 7, and 21 days post-RB-C22v inoculation. Viremia, viral replication in tissues, and oronasal shedding were reduced in animals challenged at 7 and 21 DPI. Notably BICv-specific RNA was not detected in tonsils of challenged animals. These results indicate that a carboxyl-terminal domain of E1 glycoprotein affects virulence of CSFV in swine, and they demonstrate that mutation of this domain provides the basis for a rationally designed and efficacious live-attenuated CSF vaccine.     

Differentiation of Classical Swine Fever Virus Infection from CP7_E2alf Marker Vaccination by a Multiplex Microsphere Immunoassay

Classical swine fever (CSF) is a highly contagious viral disease of pigs that has a tremendous socioeconomic impact. Vaccines are available for disease control. However, most industrialized countries are implementing stamping-out strategies to eliminate the disease and avoid trade restrictions. These restrictions can be avoided through the use of marker vaccines such as CP7_E2alf. Marker vaccines have to be accompanied by reliable and robust discriminatory assays. In this context, a multiplex microsphere immunoassay for serological differentiation of infected from vaccinated animals (DIVA) was developed to distinguish CSF virus (CSFV)-infected animals from CP7_E2alf-vaccinated animals. To this end, three viral proteins, namely, CSFV E2, CSFV E^rns, and bovine viral diarrhea virus (BVDV) E2, were produced in insect cells using a baculovirus expression system; they were used as antigens in a microsphere immunoassay, which was further evaluated by testing a large panel of pig sera and compared to a well-characterized commercial CSFV E2 antibody enzyme-linked immunosorbent assays (ELISAs) and a test version of an improved CSFV E^rns antibody ELISA. Under a cutoff median fluorescence intensity value of 5,522, the multiplex microsphere immunoassay had a sensitivity of 98.5% and a specificity of 98.9% for the detection of antibodies against CSFV E2. The microsphere immunoassay and the CSFV E^rns ELISA gave the same results for 155 out of 187 samples (82.8%) for the presence of CSFV E^rns antibodies. This novel multiplex immunoassay is a valuable tool for measuring and differentiating immune responses to vaccination and/or infection in animals.     

Phylogenetic analysis of recent isolates of classical swine fever virus from Colombia

The ability to discriminate between different classical Swine fever virus (CSFV) isolates is a prerequisite for identifying the possible origin of an outbreak. To determine the relatedness between Colombian isolates from different geographical regions, genetic sequences of the glycoprotein E2 and the 5'UTR of CSFV were amplified by PCR, sequenced and compared with reference strains of different genetic grouping. The viruses originated from classical swine fever (CSF) outbreaks in Colombia during 1998-2002. All viruses characterized belonged to genogroup 1 and were members of the subgroup 1.1. The results indicate that the outbreaks from the year 2002 are caused by a strain related to the virus CSF/Santander, isolated in 1980, suggesting that the current CSF outbreaks are the consequence of a single strain that continues to circulate in the field. For the first time, an association between isolates from outbreaks in Colombia in the 1990s was established with a virus isolate from Brazil, indicating a possible origin of the virus causing the outbreak.     

Development of a primer-probe energy transfer real-time PCR assay for improved detection of classical swine fever virus

Classical swine fever (CSF) is a contagious and devastating disease, causing serious losses in the pig industry worldwide. Vaccination of pigs with the conventional C-strain vaccine has been practised in different regions of the world in order to prevent the disease. In the control programmes of CSF, rapid detection and identification of the causing agent, classical swine fever virus (CSFV) is a crucial step. This study describes a novel real-time PCR assay based on primer-probe energy transfer (PriProET) technology for improved detection of CSFV. The assay is able to detect 20 copies of viral cDNA per reaction, showing a high sensitivity. The specificity has been evaluated by testing 57 pestiviruses, representing all species and unclassified pestiviruses. The assay has been found to be highly reproducible. Following PCR amplification, melting curve analysis allows confirmation of specific amplicons, and differentiation between wild-type CSFV and certain C-strain vaccines. This study provides a new tool for the diagnosis of CSF.